Hepatic FASN deficiency differentially affects nonalcoholic fatty liver disease and diabetes in mouse obesity models

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes are interacting comorbidities of obesity, and increased hepatic de novo lipogenesis (DNL), driven by hyperinsulinemia and carbohydrate overload, contributes to their pathogenesis. Fatty acid synthase (FASN), a key enzyme of hepatic DNL, is upregulated in association with insulin resistance. However, the therapeutic potential of targeting FASN in hepatocytes for obesity-associated metabolic diseases is unknown. Here, we show that hepatic FASN deficiency differentially affects NAFLD and diabetes depending on the etiology of obesity. Hepatocyte-specific ablation of FASN ameliorated NAFLD and diabetes in melanocortin 4 receptor–deficient mice but not in mice with diet-induced obesity. In leptin-deficient mice, FASN ablation alleviated hepatic steatosis and improved glucose tolerance but exacerbated fed hyperglycemia and liver dysfunction. The beneficial effects of hepatic FASN deficiency on NAFLD and glucose metabolism were associated with suppression of DNL and attenuation of gluconeogenesis and fatty acid oxidation, respectively. The exacerbation of fed hyperglycemia by FASN ablation in leptin-deficient mice appeared attributable to impairment of hepatic glucose uptake triggered by glycogen accumulation and citrate-mediated inhibition of glycolysis. Further investigation of the therapeutic potential of hepatic FASN inhibition for NAFLD and diabetes in humans should thus consider the etiology of obesity.


Primers for RT-qPCR analysis
. The animals were provided with the HFD or HFruD from 4 wk of age for 20 wk, or as indicated.

Metabolic tests.
For the IPGTT, mice deprived of food for 16 h were injected intraperitoneally with glucose (1.5 or 2 g/kg of BW as indicated).For the ITT, mice in the randomly fed state were injected intraperitoneally with human regular insulin (0.75, 2, or 4 U/kg of BW as indicated).For the pyruvate and glycerol tolerance tests, mice deprived of food for 16 h were injected intraperitoneally with pyruvate (2 g/kg of BW) or glycerol (3 g/kg of BW), respectively, dissolved in physiological saline.Blood glucose concentrations were measured at the indicated time points.
Metabolic measurements.Food intake and locomotor activity were determined with the use of a food intake, drinking, and locomotor activity monitoring system for mice (Shin Factory, MFD-100M and ACTIMO-100S).Oxygen consumption, carbon dioxide production, and the respiratory exchange ratio were measured every 5 min over 24 h under the resting condition with the use of an Oxymax V4.00 calorimeter (Columbus Instruments).Mice were monitored individually in these systems for 2 days (1 day of acclimation followed by 1 day of measurement).Body temperature was measured with a rectal probe attached to a digital thermometer (Physitemp Instruments, BAT-12).

Leptin supplementation. An osmotic pump (Alzet, Micro-osmotic Pump model 1002)
was implanted subcutaneously into individually caged male ob/ob;F/F or ob/ob;HKO mice at 8 to 9 wk of age.PBS vehicle or leptin at 2.0 mg/day was administered for 7 days via the pump.
Histology.Mouse liver was excised, fixed in a 10% neutral formalin solution, embedded in paraffin, sectioned at a thickness of 4 to 5 mm, and subjected to H&E or PAS staining.Frozen sections of liver tissue prepared at a thickness of 20 µm in Neg-50 Frozen Section Medium (Thermo Fisher Scientific, 6402G) were subjected to oil red O staining.
Paraffin embedding, sectioning, and H&E, PAS, and oil red O staining were performed at the Communal Laboratory, Research Institute, National Center for Global Health and Medicine.

Transduction of recombinant adenovirus vectors.
A control adenovirus encoding βgalactosidase (LacZ) and an adenovirus encoding GS were described previously (7).
Adenoviruses were injected into mice via the tail vein at a dose of 3.0 × 10 9 PFU, and the mice were analyzed in the fed or fasted state at 4 days after adenoviral transduction.
RT-qPCR analysis.Total RNA was extracted from pulverized liver with the use of an RNeasy Mini Kit and RNase-Free DNase Set (Qiagen: 74104 and 79254, respectively) or a NucleoSpin RNA Kit (Macherey Nagel, 740955).The RNA was subjected to RT with random primers and the use of a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813).The resulting cDNA was subjected to qPCR analysis in triplicate with the use of a StepOnePlus Real-Time PCR System and Power UP SYBR Green Master Mix (Applied Biosystems, A25742).Relative mRNA abundance was calculated by the standard curve method and with normalization according to the corresponding amount of 18S rRNA.
Primer sequences for qPCR are listed in Supplemental Table 5 and were described previously (8).
Metabolite measurement.Frozen liver tissue (~50 mg) was transferred to 1.5 mL of 50% acetonitrile solution in deionized water containing internal standards (Solution ID 304-1002; Human Metabolome Technologies, Yamagata, Japan) at 0ºC in order to inactivate enzymes.The tissue was homogenized three times at 1500 rpm for 120 s with a Micro Smash MS100R device (Tomy Digital Biology), and the homogenate was centrifuged at 2300 × g and 4ºC for 5 min.A portion (800 µL) of the resulting supernatant was centrifugally filtered through a Millipore 5-kDa cutoff filter (Merck Millipore) at 9100 × g and 4ºC for 120 min to remove proteins.The filtrate was evaporated, and the residue was resuspended in 50 µL of deionized water for mass spectrometric analysis.Metabolome analysis was performed through a facility service at Human Metabolome Technologies by capillary electrophoresis-TOF mass spectrometry for cation analysis and capillary electrophoresis-tandem mass spectrometry for anion analysis.Data are provided in Supplemental Tables 1 and 2. Hepatic citrate content was measured with 20 mg of liver tissue and a Citrate Assay Kit (Sigma-Aldrich, MAK057).In brief, the tissue was homogenized in Citrate Assay Buffer and the homogenate was centrifuged at 15,000 × g and 4ºC for 10 min to remove insoluble material.
The resulting supernatant was depleted of protein with a Vivaspin 500 10,000 MWCO device (Sartorius, VS0101) and was then analyzed in triplicate, with the results expressed as nanomoles of citrate per gram of liver weight.
Measurement of hepatic glycogen, triglyceride, and cholesterol levels.Hepatic glycogen content was measured with 50 mg of liver tissue and a Glycogen Colorimetric Assay Kit (BioVision, K646-100).For determination of cholesterol and triglyceride levels, lipids were extracted from 40 to 50 mg of liver tissue with chloroform-methanol (2:1, vol/vol) according to the Folch method (9).A portion of the organic phase was dried, resuspended in 2-propanol, and analyzed for triglyceride or cholesterol with a Triglyceride Etest or Cholesterol E-test (Fujifilm Wako Pure Chemical), respectively.Samples were analyzed in triplicate and the results expressed as milligrams of lipid per gram of liver weight.
Determination of tissue glucose uptake, and liver PFK and glycogen phosphorylase activity.Mice that had been deprived of food for 16 h were injected intraperitoneally with a mixture of glucose and 2-DG at 0.5 and 1.5 g per kilogram of BW, respectively, dissolved in PBS.Liver and gastrocnemius muscle were excised rapidly at 30 min after 2-DG administration, and 2-deoxyglucose 6-phosphate levels in the tissue were determined with a 2-Deoxyglucose Uptake Measurement Kit (Cosmo Bio, MBR-PMG-K01).Hepatic PFK activity was measured with 40 mg of frozen liver tissue and a PFK Activity Colorimetric Assay Kit (BioVision, K776-100).Hepatic glycogen phosphorylase activity was measured with 50 mg of frozen liver tissue and a Glycogen Phosphorylase Assay Kit (Colorimetric) (Abcam, ab273271).
In vivo DNL assay.Mice were injected intraperitoneally with 35 µL per gram of BW of deuterated water ( 2 H2O) (Sigma-Aldrich, 151882) or H2O containing 0.9% NaCl and were maintained for 24 h with drinking water consisting of 8% enriched 2 H2O or H2O.They were deprived of food for the final 6 h of this labeling period and then killed by cervical dislocation for collection of plasma and the liver.
For 2 H enrichment analysis of liver palmitate, gas chromatography-mass spectrometry (GC-MS) analysis was performed as previously described (10), with minor modifications.Frozen liver tissue (25-50 mg) was pulverized with an automatic cryogenic pulverizer (Tokken), and Methylation Reagent A from a Fatty Acid Methylation Kit (Nacalai Tesque, 06482-04) and 1 µg of C23:0 fatty acid (Sigma-Aldrich, T6543) as an internal standard were directly added to the pulverized tissue.Total fatty acids (free and esterified) were methylated with the use of the Fatty Acid Methylation Kit, and the resulting fatty acid methyl esters (FAMEs) were purified with a Fatty Acid Methyl Ester Purification Kit (Nacalai Tesque, 06483-94).The FAME samples were evaporated, reconstituted in dichloromethane, and analyzed with a GCMS-QP2010 Ultra system (Shimadzu) equipped with a FAMEWAX fused silica capillary column (length of 30 m with an internal diameter of 0.25 mm and a film thickness of 0.25 µm) (Restek, 12497).Each sample (1 µL) was injected in splitless mode, with helium as the carrier gas at a flow rate of 45 cm/s (linear velocity).
The injection port temperature was set at 250°C.The oven temperature was set to 40°C and maintained for 2 min before being increased to 140°, 200°, and 240°C at a rate of 20°, 11°, and 3°C/min, respectively, and it was then maintained at 240°C for 10 min.The interface and ion source temperatures were maintained at 250° and 200°C, respectively.The mass spectrometer was operated in electron impact ionization mode with an ionization energy of 70 eV.Selected ion monitoring was performed for specific ions.The ions monitored for methyl esterified palmitate with deuterium incorporation were those with a mass/charge (m/z) ratio of 270, 271, 272, 273, 274, 275, 276, 277, and 278.An m/z of 368 was used to monitor C23:0 fatty acid.Mass isotopomer (m) distribution was determined by weighted multiple linear regression analysis with the use of the m/z spectrum of methyl esterified palmitate from liver samples of H2O-loaded mice to remove anything other than 2 H from 2 H2O that might add mass to palmitic acid (such as a contribution of the derivatizing agent or the natural abundance of 13 C, 2 H, and 18 O) (11).The calculated mass isotopomer distribution was expressed as molar fractions (m0, m1, m2, etc., corresponding to the fractions of molecules containing 0, 1, 2, etc. 2 H substitutions, respectively).The relative amount of total tissue palmitate was determined by normalization with the AUC of C23:0 fatty acid.
The 2 H enrichment of water from plasma samples and standards was determined by deuterium acetone exchange (12).A 10-µL volume of sample or standard was mixed with 2 µL of 10 M NaOH and 5 µL of acetone in a GC vial and was incubated at room temperature for 4 h before analysis.Acetone was analyzed with the GCMS-QP2010 Ultra instrument equipped with a DB-17MS column (length of 15 m with an internal diameter of 0.25 mm and a film thickness of 0.15 µm) (Agilent, 122-4711).A portion (1 µL) of headspace acetone gas from the GC vial was injected in splitless mode, with helium as the carrier gas at a flow rate of 54.1 cm/s (linear velocity).The injection port temperature was set at 170°C.The oven temperature was set and held at 170°C for 2 min.The interface and ion source temperatures were maintained at 250° and 200°C, respectively.The mass spectrometer was operated in electron impact ionization mode with an ionization energy of 70 eV.Selected ion monitoring was adopted for specific ions.The ions monitored were those with an m/z of 58, 59, and 60.
The AUC was used to calculate the 2 H enrichment of the samples by calibration with standards with a known 2 H mole fraction prepared by mixing H2O and 99.9% 2 H2O.
The fraction of newly synthesized fatty acids (FNS) was calculated as previously described (10)

Supplemental Figure 1 .Supplemental Figure 2 .Supplemental Figure 3 .Supplemental Figure 4 .Supplemental Figure 5 .Supplemental Figure 6 .
Targeting of Fasn and generation of HKO mice.(A) Targeting vector, WT allele, and loxP-containing Fasn alleles before and after recombination.The post-Cre allele represents the Fasn allele after deletion of exons 4 to 10. DT-A and NEO denote a diphtheria toxin-A cassette for negative selection and a neomycin resistance cassette for positive selection, respectively.Gray boxes and closed arrowheads represent exons and loxP sites, respectively, and arrows A to C indicate genotyping primers (primer A, 5′-CAGCTAT-GAAGCAATTGTGGATGG-3′; primer B, 5′-AGTGGGACAGGGTTGTTGCCAAGC-3′; primer C, 5′-GAAGTCAGTAGGTCGATGAGGGC-3′).Probes A, N, and J are for Southern hybridization.B, BamHI site; S, SmaI site; K, KpnI site; X, XhoI site; E, EcoRV site.(B) Southern blot analysis of DNA from embryonic stem cells harboring a Fasn allele targeted by the targeting vector (initial targeted allele) and a WT allele.Genomic DNA was digested with BamHI and hybridized with external probe A (DNA fragment between two SmaI sites in the 5′ region of Fasn) or probe N (NEO cassette), or was digested with KpnI and hybridized with external probe J (DNA fragment between EcoRV and KpnI sites in the 3′ region of Fasn).(C) RT-qPCR analysis of Fasn mRNA in the liver of 10-wk-old WT, F/F (control), and HKO mice.Data are means + SEM (n = 5 mice).**P < 0.01 (one-way ANOVA followed by Tukey's multiple comparison test).(D) Immunoblot (IB) analysis of FASN and α-tubulin (Tub, loading control) in various tissues of 10-wk-old WT, F/F, and HKO mice.WAT, white adipose tissue; BAT, brown adipose tissue.Metabolic characteristics of 12-wk-old NCD-fed HKO mice.(A-C) BW (A) as well as blood glucose (B) and plasma insulin (C) concentrations in the fed state for HKO mice and F/F littermate controls (n = 8).(D and E) An IPGTT (2 g of glucose per kilogram of BW) (D) and ITT (0.75 U of human regular insulin per kilogram of BW) (E) for HKO mice and littermate controls (n = 4 or 5).All data are means + SEM for the indicated numbers (n) of mice.Metabolic characteristics of 24-wk-old HFD-fed HKO mice.(A-C) BW (n = 10) (A), food intake over 24 h (n = 6) (B), and weight of the liver, inguinal fat, and epididymal fat (n = 6) (C) for F/F and HKO mice maintained on an HFD.(D and E) Blood glucose (D) and plasma insulin (E) concentrations in the fed state for HKO mice and littermate controls (n = 10).(F and G) An IPGTT (1.5 g of glucose per kilogram of BW) (F) of human regular insulin per kilogram of BW) (G) for HKO mice and littermate controls (n = 5 or 6).(H) Hepatic triglyceride and cholesterol levels in the fed state for HKO mice and littermate controls (n = 6).All data are means + SEM for the indicated numbers of mice.**P < 0.01 (two-tailed Student's t test).Effects of hepatic FASN deficiency on energy metabolism and hepatic insulin signaling in NCD-fed ob/ob mice.(A and B) Food intake (A) and locomotor activity (B) over 24 h for ob/ob;F/F and ob/ob;HKO mice at 10 wk of age (n = 6).(C) Body temperature (n = 8) and RT-qPCR analysis of Ucp1 mRNA in brown adipose tissue (n = 5) for mice at 10 wk of age.(D-F) Oxygen consumption (VO2) (D) and carbon dioxide ob/ob;F/F ob/ob;HKO ob/ob;F/F ob/ob;HKO ob/ob;F/F ob/ob;HKO ob/ob;F/F ob/ob;HKO production (VCO2) (E) over 24 h as well as the respiratory exchange ratio (RER) in the dark and light phases (F) for mice at 8-to 12-wk of age (n = 5 or 6).(G) Immunoblot analysis of Ser 473 -phosphorylated and total forms of Akt in the liver of 10-wk-old mice in the fasted and fed states.(H) Effects of insulin on IRb and Akt phosphorylation in the liver of 8-wk-old mice.Mice deprived of food overnight were injected intravenously with insulin (5 U/kg) or vehicle (-), 2 min after which the liver was isolated, lysed, and subjected to immunoprecipitation (IP) with antibodies to IRb.The resulting precipitates were subjected to immunoblot analysis with antibodies to phosphotyrosine (PY20) or to IRb.Alternatively, the liver lysates were subjected directly to immunoblot analysis with antibodies to Ser 473phosphorylated or total forms of Akt.Each lane in G and H corresponds to one mouse.All quantitative data are means + SEM for the indicated numbers of mice.*P < 0.05, **P < 0.01 compared with ob/ob;F/F mice or as indicated (two-tailed Student's t test).Effects of an HFruD on the metabolic phenotype of 24-wk-old ob/ob;HKO mice.(A) Food intake over 24 h for ob/ob;F/F and ob/ob;HKO mice maintained on an HFruD (n = 7).(B and C) Plasma fructose (n = 4) (B) and insulin (n = 5) (C) concentrations in the fed state for HFruD-fed mice.(D) Hepatic glycogen content (mg/liver) in the fed state for HFruD-fed mice (n = 4).(E) Immunoblot analysis of phosphorylated and total forms of GS and GSK-3α/β in the liver of HFruD-fed mice in the fed state (n = 3).(F) Hepatic glycogen phosphorylase activity in the fed or fasted state for HFruD-fed mice (n = 3 or 4).All quantitative data are means + SEM for the indicated numbers of mice.*P < 0.05, /liver) HFruD ob/ob;F/F ob/ob;HKO D Phospho-GSK-3α/β (Ser 21/9 ) GSK-3α/β FASN GS Phospho-GS (Ser 641 ) Tub ob/ob;F/F ob/ob;HKO IB: < 0.01 [two-tailed Student's t test (D) or one-way ANOVA followed by Tukey's multiple comparison test (F)].Hepatic expression of lipogenic enzymes in F/F, HKO, Mc4rKO;F/F, and Mc4rKO;HKO mice as well as of SREBP1c and ChREBP target genes in F/F, ob/ob;F/F, and Mc4rKO;F/F mice.(A) Immunoblot analysis of FASN, ACC, and ACLY in the liver of NCD-fed 10-wk-old F/F and HKO mice and 16-to 20-wk-old Mc4rKO;F/F and Mc4rKO;HKO mice in the fed state.Each lane corresponds to one mouse.(B) RT-qPCR analysis of target genes of SREBP1c, ChREBP, or both transcription factors in the liver of 10-wk-old F/F and ob/ob;F/F mice as well as of 22-to 24-wk-old Mc4rKO;F/F mice in the fed state.Elovl6, elongation of long-chain fatty acids family member 6 gene; Pnpla3, patatin-like phospholipase domain-containing protein 3 gene; Scd1, stearoyl-CoA desaturase 1 gene.Data are means + SEM for six mice.*P < 0.05, **P < 0.01 (one-way ANOVA followed by Tukey's multiple comparison test).
Ucp1 5'-GATCCGAGGGCCTCACTAAACC-3' 5'-AATTCCCAGTAAGTGCGGGTCA-3' Rn18s of these strains were also observed at the expected Mendelian frequency.We maintained Mc4rKO;F/F and Mc4rKO;HKO mice on the C57BL/6J background and used male mice for the present study.Mice were provided ad libitum access to either an NCD (CLEA Japan, CE-2), HFD (Research Diets, D12492), or HFruD (Research Diets, D00111301).The composition of the diets is shown in Supplemental Table In brief, FNS is determined by the following equation: FNS = ME/(N ´ p), where ME is the average number of 2 H atoms incorporated per molecule [ME = (m1 ´ 1) + (m2 ´ 2) + (m3 ´ 3) +…] and p is the 2 H enrichment of water calculated as above.N represents the number of exchangeable hydrogens and was calculated by the following equation: m2/m1 = [(N -1)/2] ´ [p/(1 -p)].The relative amount of newly synthesized palmitate was determined by multiplying FNS by the relative amount of total tissue palmitate and then normalizing by tissue weight.

Hepatic metabolite abundance (nmol/g) in ob/ob;F/F and ob/ob;HKO mice in the fasted state
Data are means ± SEM (n = 4 mice).a, Analyzed by Welch's t test.b, Analyzed by Student's t test.

Supplemental Table 2 Hepatic metabolite abundance in ob/ob;F/F and ob/ob;HKO mice in the fed state
Data are means ± SEM (n = 3-6 mice)and are expressed as nmol/g or *relative area/mg.N/A, not available, a, Analyzed by Student's t test.b, Analyzed by Welch's t test.